Leukotriene receptor

ABSTRACT

The present invention provides an isolated mammalian leukotriene receptor, isolated or recombinant nucleic acids and recombinant vectors encoding the same, host cells comprising the nucleic acids and vectors, and methods of making the receptor using the host cells. This invention further provides antibodies and antigen binding fragments thereof which specifically bind to the receptor and are useful for treating medical conditions caused or mediated by leukotriene. Also provided are screening methods for identifying specific agonists and antagonists of the mammalian leukotriene receptor.

This is a division of application Ser. No. 09/400,622, filed Sep. 20,1999 now U.S. Pat. No. 6,465,212 B1 which is herein incorporated byreference.

TECHNICAL FIELD

The present invention relates to mammalian leukotriene receptors. Moreparticularly, it relates to human leukotriene receptors, isolatednucleic acids and recombinant vectors encoding the receptors, to methodsof making the receptors, to methods of making fragments or fusionproteins of the receptors using recombinant DNA methodology or chemicalsynthesis, and to methods of using the receptors in screening systems toidentify inhibitors and activators of the receptors useful for thetreatment of various diseases.

BACKGROUND OF THE INVENTION

Leukotrienes are products of eicosanoid metabolism and are implicated ina number of medical conditions, including inflammation, asthma, allergy,glomerulonephritis, neuroendocrine dysfunctions, AIDS, arthritis, boweldisease, psoriasis, diabetes, obesity, atherosclerosis, bacterialinfection, etc. There are multiple classes of leukotrienes, e.g. classA, class B, class C, class D, class E and class F. Leukotrienes regulatethe intensity and duration of immune responses and are involved incell-to-cell communication. Leukotrienes are also involved in leukocytemigration and branchovasoconstriction. As established by radioligandbinding as well as physiological assays, it appears that there aredifferent types of receptors for leukotrienes. The molecularcharacteristics of leukotriene receptors were unknown until recently,when a leukotriene B4 receptor was cloned (Yokomizo et al., Nature,387:620-624 (1997)).

In view of the important role that leukotrienes play in manyphysiological processes and medical conditions, there is a need formaterials and methods useful for the identification of agonists andantagonists of leukotriene receptors.

SUMMARY OF THE INVENTION

The present invention fills the foregoing need by providing suchmaterials and methods. More particularly, this invention provides anovel mammalian leukotriene receptor, isolated nucleic acids orrecombinant nucleic acids encoding the receptor, and recombinant vectorsand host cells comprising such nucleic acids. The leukotriene receptorcan be actively expressed in mammalian cells where it displays activeligand binding and positive intracellular signaling upon ligandactivation. This novel receptor has high affinity for ligands such asleukotriene B4, leukotriene B5, 15(5)-OH-5z, 8z, 11z,13e)-eicosatetraenoic acid, and lipoxin A4. This invention furtherprovides methods for the discovery of selective agonists and antagonistsof the receptor that may be useful in the treatment and management of avariety of diseases including, for example, inflammation, asthma,allergy, glomerulonephritis, neuroendocrine dysfunctions, alcoholicliver diseases, hepatitis, atropic liver regeneration, AIDS, arthritis,bowel disease, psoriasis, diabetes, obesity, atherosclerosis, andbacterial infection.

The isolated or recombinant nucleic acids of the present invention areselected from the group consisting of:

(a) A nucleic acid encoding a mammalian leukotriene receptor comprisingan amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof;

(b) A nucleic acid that hybridizes under moderately stringent conditionsto the nucleic acid of (a) and encodes a polypeptide that (i) bindsleukotriene and (ii) is at least 80% identical to a receptor encoded bythe nucleic acid of (a); and

(c) A nucleic acid that, due to the degeneracy of the genetic code,encodes a mammalian leukotriene receptor encoded by a nucleic acid of(a) or (b).

This invention further provides methods of making a polypeptidecomprising culturing a host cell comprising a nucleic acid encoding amammalian leukotriene receptor comprising an amino acid sequence definedby SEQ ID NO: 2 or a subsequence thereof, under conditions in which thenucleic acid is expressed. In some embodiments, the method furthercomprises isolation of the polypeptide from the culture.

This invention also provides a recombinant nucleic acid comprising asequence having at least about 70% identity over a stretch of at leastabout 30 nucleotides to the nucleic acid sequence of SEQ ID NO: 1,useful, e.g., as a probe or PCR primer for a related gene. Anotherembodiment further includes a polypeptide comprising at least about 60%identity over a stretch of at least about 20 amino acids to the aminoacid sequence of SEQ ID NO: 2.

This invention also provides polypeptides comprising a fragment of apolypeptide having an amino acid sequence corresponding to the sequenceof at least about 8 contiguous residues of the amino acid sequence ofSEQ ID NO: 2. Preferably, the polypeptides comprise at least about 12,more preferably at least about 20, and most preferably at least about 30such residues.

Still further, this invention provides fusion proteins comprising apolypeptide defined by SEQ ID NO: 2 or a fragment therefrom covalentlylinked to a fusion partner.

The present invention also provides antibodies, both polyclonal andmonoclonal, that specifically bind to one or more of the leukotrienereceptors or to polypeptides therefrom, and anti-idiotypic antibodies,both monoclonal and polyclonal, which specifically bind to the foregoingantibodies.

This invention further provides a method for producing a mammalianleukotriene receptor comprising culturing a host cell comprising anucleic acid encoding a mammalian leukotriene receptor comprising anamino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof,under conditions in which the nucleic acid is expressed. In oneembodiment the receptor is isolated from the culture.

The present invention also provides a method for identifying aleukotriene agonist or antagonist comprising:

-   -   (a) Contacting a mammalian leukotriene receptor having an amino        acid sequence defined by SEQ ID NO: 2 or a subsequence thereof,        in the presence of a known amount of labeled leukotriene or        surrogate with a sample to be tested for the presence of a        leukotriene agonist or antagonist; and    -   (b) Measuring the amount of labeled leukotriene specifically        bound to the receptor;        whereby a leukotriene agonist or antagonist in the sample is        identified by measuring substantially reduced binding of the        labeled leukotriene to the leukotriene receptor, compared to        what would be measured in the absence of such agonist or        antagonist.

In a preferred embodiment, membranes isolated from mammalian cellscomprising a nucleic acid encoding the leukotriene receptor are used asthe source of the receptor.

The present invention also provides a method for identifying an agonistor antagonist of a mammalian leukotriene receptor comprising:

(a) contacting cells expressing a mammalian leukotriene receptorcomprising an amino acid sequence defined by SEQ ID NO: 2 or aconservative or allelic variant thereof, in the presence of a knownamount of leukotriene or surrogate thereof with a sample to be testedfor the presence of a mammalian leukotriene agonist or antagonist; and

(b) measuring at least one cellular function modulated by the binding ofa ligand to said receptor present in the cells;

whereby a mammalian leukotriene receptor agonist or antagonist in thesample is identified by measuring its effect on said cellular functioncompared to what would be measured in the absence of such agonist orantagonist.

Examples of cellular functions modulated by the binding of a ligand to amammalian leukotriene receptor include: intracellular second messengerpathways activated via the leukotriene receptors (e.g., cyclicAMP,calcium, inositol phosphate and MAP kinase), changes in cell growthrate, secretion of hormones, receptor-stimulated Ca²⁺⁺ mobilization,mitogenic effects etc.,

This invention still further provides a method for treatingleukotriene-mediated medical conditions comprising administering to amammal afflicted with a medical condition caused or mediated byleukotriene, an effective amount of an agonist or antagonist of theleukotriene receptor that specifically binds to a mammalian leukotrienereceptor having an amino acid sequence defined by SEQ ID NO: 2, or asubsequence thereof, and pharmaceutical compositions comprising one ormore of such agonist or antagonist and a pharmaceutically acceptablecarrier. Preferably, the mammal is a human being.

This invention also provides anti-sense oligonucleotides capable ofspecifically hybridizing to mRNA encoding a mammalian leukotrienereceptor having an amino acid sequence defined by SEQ ID NO: 2 or asubsequence thereof so as to prevent translation of the mRNA.Additionally, this invention provides anti-sense oligonucleotidescapable of specifically hybridizing to the genomic DNA molecule encodinga mammalian leukotriene receptor having an amino acid sequence definedby SEQ ID NO: 2 or a subsequence thereof.

This invention further provides a pharmaceutical composition comprising:

(a) An amount of an oligonucleotide effective to reduce activity ofhuman leukotriene receptor by passing through a cell membrane andbinding specifically with DNA or mRNA encoding human leukotrienereceptor in the cell so as to prevent its transcription or translation;and

(b) A pharmaceutically acceptable carrier capable of passing through acell membrane. In an embodiment, the oligonucleotide is coupled to asubstance that inactivates mRNA.

In another embodiment, the substance that inactivates mRNA is aribozyme.

DESCRIPTION OF THE INVENTION

All references cited herein are hereby incorporated herein in theirentirety by reference.

Leukotriene Receptor Characterization

The nucleotide sequence of the complete open reading frame and thecorresponding amino acid sequence of the novel human leukotrienereceptor of this invention are defined by SEQ ID NO: 1 and SEQ ID NO: 2,respectively. The cloned receptor resembles a member of the G-proteincoupled receptor super-family that contains a 7-transmembrane domain.Furthermore, this receptor shares high homology at both the nucleotideand amino acid sequence levels with the previously described leukotrienereceptor (Yokomizo et al., Nature, 387:620-624 (1997)). The clonedreceptor is able to bind ligands (for example, leukotriene B and lipoxinA), as demonstrated with radioligand saturation and competition assays.Leukotriene is also capable of activating the cloned receptor resultingin intracellular responses, as shown by measurement of intracellularCa²⁺ flux.

As used herein, the term “ligand” is defined to mean any moleculecapable of specifically binding to the mammalian leukotriene receptorsof the invention. Thus leukotriene itself is a ligand, as are agonistsand antagonists that may compete with leukotriene for specific bindingto the receptors.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell's post-translationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same post-translational glycosylations asmammalian cells do and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins having thenative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

The term “polypeptide” encompasses all such modifications, particularlythose that are present in polypeptides synthesized by expressing apolynucleotide in a host cell.

“Variant(s)”, as the term is used herein, are polynucleotides orpolypeptides that differ from a reference polynucleotide or polypeptiderespectively. Variants in this sense are described below and elsewherein the present disclosure in greater detail. (1) A polynucleotide thatdiffers in nucleotide sequence from another, reference polynucleotide.Changes in the nucleotide sequence of the variant may be silent, i.e.they may not alter the amino acids encoded by the polynucleotide. Wherealterations are limited to silent changes of this type a variant willencode a polypeptide with the same amino acid sequence as the referencepolypeptide. Changes in the nucleotide sequence of the variant may alterthe amino acid sequence of a polypeptide encoded by the referencepolynucleotide. Such nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. (2) Apolypeptide that differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more substitutions, additions, deletions,fusions and truncations, which may be present in any combination. (3) Avariant may also be a fragment of a polynucleotide or polypeptide of theinvention that differs from a reference polynucleotide or polypeptidesequence by being shorter than the reference sequence, such as by aterminal or internal deletion. A variant of a polypeptide of theinvention also includes a polypeptide which retains essentially the samebiological function or activity as such polypeptide, e.g., pro-proteinswhich can be activated by cleavage of the pro-protein portion to producean active mature polypeptide. (4) A variant may also be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not beencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a pro-protein sequence. (5) A variant of thepolynucleotide or polypeptide may be a naturally occurring variant suchas a naturally occurring allelic variant, or it may be a variant that isnot known to occur naturally. Such non-naturally occurring variants orthe polynucleotide may be made by mutagenesis techniques, includingthose applied to polynucleotides, cells or organisms, or may be made byrecombinant means. Among polynucleotide variants in this regard arevariants that differ from the aforementioned polynucleotides bynucleotide substitutions, deletions or additions. The substitutions,deletions or additions may involve one or more nucleotides. The variantsmay be altered in coding or non-coding regions or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. All such variants definedabove are deemed to be within the scope of those skilled in the art fromthe teachings herein and from the art.

The present invention also encompasses fragments, analogs and physicalvariants of the disclosed leukotriene receptor. As used herein, the term“polypeptide” or “peptide” means a fragment or segment, e.g., of amammalian leukotriene receptor having an amino acid sequence defined bySEQ ID NO: 2 which comprises a subsequence of the complete amino acidsequence of the receptor containing at least about 8, preferably atleast about 12, more preferably at least about 20, and most preferablyat least about 30 or more contiguous amino acid residues, up to andincluding the total number of residues in the complete receptor.

The polypeptides of the invention can comprise any part of the completesequence of such a receptor. Thus, although they could be produced byproteolytic cleavage of an intact receptor, they can also be made bychemical synthesis or by the application of recombinant DNA technologyand are not limited to polypeptides delineated by proteolytic cleavagesites. The polypeptides, either alone or cross-linked or conjugated to acarrier molecule to render them more immunogenic, are useful as antigensto elicit the production of antibodies. The antibodies can be used,e.g., in immunoassays of the intact receptors, for immunoaffinitypurification, etc.

The term “analog(s)” means a mammalian leukotriene receptor of theinvention which has been modified by deletion, addition, modification orsubstitution of one or more amino acid residues in the wild-typereceptor. It encompasses allelic and polymorphic variants, and alsomuteins and fusion proteins which comprise all or a significant part ofsuch a mammalian leukotriene receptor, e.g., covalently linked via aside-chain group or terminal residue to a different protein, polypeptideor moiety (fusion partner).

Some amino acid substitutions are preferably “conservative”, withresidues replaced with physically or chemically similar residues, suchas Gly/Ala, Asp/Glu, Val/Ile/Leu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr.Analogs having such conservative substitutions typically retainsubstantial leukotriene binding activity. Other analogs, which havenon-conservative substitutions such as Asn/Glu, Val/Tyr and His/Glu, maysubstantially lack such activity. Nevertheless, such analogs are usefulbecause they can be used as antigens to elicit production of antibodiesin an immunologically competent host. Because these analogs retain manyof the epitopes (antigenic determinants) of the wild-type receptors fromwhich they are derived, many antibodies produced against them can alsobind to the active-conformation or denatured wild-type receptors.Accordingly, such antibodies can also be used, e.g., for theimmunopurification or immunoassay of the wild-type receptors.

Some analogs are truncated variants in which residues have beensuccessively deleted from the amino- and/or carboxyl-termini, whilesubstantially retaining the characteristic ligand binding activity.

Modifications of amino acid residues may include but are not limited toaliphatic esters or amides of the carboxyl terminus or of residuescontaining carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino-terminalamino acid or amino-group containing residues, e.g., lysine or arginine.

Other analogs are mammalian leukotriene receptors containingmodifications, such as incorporation of unnatural amino acid residues,or phosphorylated amino acid residues such as phosphotyrosine,phosphoserine or phosphothreonine residues. Other potentialmodifications include sulfonation, biotinylation, or the addition ofother moieties, particularly those that have molecular shapes similar tophosphate groups.

Analogs of the mammalian leukotriene receptors can be prepared bychemical synthesis or by using site-directed mutagenesis [Gillman etal., Gene 8:81 (1979); Roberts et al., Nature, 328:731 (1987) or Innis(Ed.), 1990, PCR Protocols: A Guide to Methods and Applications,Academic Press, New York, N.Y.] or the polymerase chain reaction method[PCR; Saiki et al., Science 239:487 (1988)], as exemplified by Daughertyet al. [Nucleic Acids Res. 19:2471 (1991)] to modify nucleic acidsencoding the complete receptors. Adding epitope tags for purification ordetection of recombinant products is envisioned.

General techniques for nucleic acid manipulation and expression that canbe used to make the analogs are described generally, e.g., in Sambrook,et al., Molecular Cloning: A Laboratory Manual (2d ed.), 1989, Vols.1-3, Cold Spring Harbor Laboratory. Techniques for the synthesis ofpolypeptides are described, for example, in Merrifield, J. Amer. Chem.Soc. 85:2149 (1963); Merrifield, Science 232:341 (1986); and Atherton etal., Solid Phase Peptide Synthesis: A Practical Approach, 1989, IRLPress, Oxford.

Still other analogs are prepared by the use of agents known in the artfor their usefulness in cross-linking proteins through reactive sidegroups. Preferred derivatization sites with cross-linking agents arefree amino groups, carbohydrate moieties and cysteine residues.

Substantial retention of ligand binding activity by the foregoinganalogs of the mammalian leukotriene receptors typically entailsretention of at least about 50%, preferably at least about 75%, morepreferably at least about 80%, and most preferably at least about 90% ofthe leukotriene binding activity and/or specificity of the correspondingwild-type receptor.

Nucleic Acids and Expression Vectors

As used herein, the term “isolated nucleic acid” means a nucleic acidsuch as an RNA or DNA molecule, or a mixed polymer, which issubstantially separated from other components that are normally found incells or in recombinant DNA expression systems. These components includebut are not limited to ribosomes, polymerases, serum components, andflanking genomic sequences. The term thus embraces a nucleic acid thathas been removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules but may, in some embodiments, contain minor heterogeneity.Such heterogeneity is typically found at the ends of nucleic acid codingsequences or in regions not critical to a desired biological function oractivity.

A “recombinant nucleic acid” is defined either by its method ofproduction or structure. Some recombinant nucleic acids are thus made bythe use of recombinant DNA techniques which involve human intervention,either in manipulation or selection. Others are made by fusing twofragments that are not naturally contiguous to each other. Engineeredvectors are encompassed, as well as nucleic acids comprising sequencesderived using any synthetic oligonucleotide process.

For example, a wild-type codon may be replaced with a redundant codonencoding the same amino acid residue or a conservative substitution,while at the same time introducing or removing a nucleic acid sequencerecognition site. Similarly, nucleic acid segments encoding desiredfunctions may be fused to generate a single genetic entity encoding adesired combination of functions not found together in nature. Althoughrestriction enzyme recognition sites are often the targets of suchartificial manipulations, other site-specific targets, e.g., promoters,DNA replication sites, regulation sequences, control sequences, or otheruseful features may be incorporated by design. Sequences encodingepitope tags for detection or purification as described above may alsobe incorporated.

A nucleic acid “fragment” is defined herein as a nucleotide sequencecomprising at least about 17, generally at least about 25, preferably atleast about 35, more preferably at least about 45, and most preferablyat least about 55 or more contiguous nucleotides.

This invention further encompasses recombinant DNA molecules andfragments having sequences that are identical or highly homologous tothose described herein. The nucleic acids of the invention may beoperably linked to DNA segments that control transcription, translation,and DNA replication.

“Identity”, as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide. “Identity” and “similarity” can be readily calculated byknown methods, including but not limited to those described in(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12 (1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J.Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.215:403-410 (1990). The well-known Smith Waterman algorithm may also beused to determine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing:

-   1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)    Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.    Natl. Acad. Sci. USA. 89:10915-10919 (1992)    Gap Penalty: 12    Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, located in Madison, Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

-   1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)    Comparison matrix: matches=+10, mismatch=0    Gap Penalty: 50    Gap Length Penalty: 3    Available as the Gap program from Genetics Computer Group, located    in Madison, Wis. Given above are the default parameters for nucleic    acid comparisons.

Preferred polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO: 1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO: 1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO: 1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO: 1, or: n_(n) x_(n)−(x_(n) y), wherein n_(n) is the number ofnucleotide alterations, x_(n) is the number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and isthe symbol for the multiplication operator, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n). Alterations of a polynucleotide sequenceencoding the polypeptide of SEQ ID NO: 2 may create nonsense, missenseor frameshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO: 2, that is itmay be 100% identical, or it may include up to a certain integer numberof amino acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected form the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of amino acids in SEQ ID NO: 2 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of amino acids in SEQ ID NO: 2, orn_(n)=x_(n)−(x_(n) y), wherein n_(n) is the number of amino acidalterations, x_(n) is the total number of amino acids in SEQ ID NO: 2, yis, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., is thesymbol for the multiplication operator, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n).

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50, 60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO: 2, wherein said polypeptide sequence may be identical to thereference sequence of SEQ ID NO: 2 or may include up to a certaininteger number of amino acid alterations as compared to the referencesequence, wherein said alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino-or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence, and wherein said number of aminoacid alterations is determined by multiplying the total number of aminoacids in SEQ ID NO: 2 by the integer defining the percent identitydivided by 100 and then subtraction that product from said total numberof amino acids in SEQ ID NO: 2, or: n_(a) x_(a)−(x_(a) y), wherein n_(a)is the number of amino acid alterations, x_(a) is the total number ofamino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and is the symbol for the multiplication operator,and wherein any non-integer product of x_(a) and y is rounded down tothe nearest integer prior to subtracting it from x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO: 2, that is it maybe 100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino-orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO: 2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO: 2, or n_(a)=x_(a) (x_(a) y), wherein n_(a) is the number of aminoacid alterations, x_(a) is the total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., andis the symbol for the multiplication operator, and wherein anynon-integer product of x_(a) and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

The term “homology”, as it is used herein, embraces both identity andsimilarity.

Some of the physical variants have substantial amino acid sequencehomology with the amino acid sequences of the mammalian leukotrienereceptors or polypeptides. In this invention, amino acid sequencehomology, or sequence identity is determined by optimizing residuematches and, if necessary, by introducing gaps as required. Homologousamino acid sequences are typically intended to include natural allelic,polymorphic and interspecies variations in each respective sequence.

Typical homologous proteins or peptides will have from 25-100% homology(if gaps can be introduced) to 50-100% homology (if conservativesubstitutions are included), with the amino acid sequence of theleukotriene receptors. Primate species receptors are of particularinterest.

Observed homologies will typically be at least about 35%, preferably atleast about 50%, more preferably at least about 75%, and most preferablyat least about 80% or more. See Needleham et al., J. Mol. Biol.48:443-453 (1970); Sankoff et al. in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison, 1983,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif., and the University of WisconsinGenetics Computer Group, Madison, Wis.

Glycosylation variants include, e.g., analogs made by modifyingglycosylation patterns during synthesis and processing in variousalternative eukaryotic host expression systems, or during furtherprocessing steps. Particularly preferred methods for producingglycosylation modifications include exposing the mammalian leukotrienereceptors to glycosylating enzymes derived from cells that normallycarry out such processing, such as mammalian glycosylation enzymes.Alternatively, deglycosylation enzymes can be used to removecarbohydrates attached during production in eukaryotic expressionsystems.

“Homologous nucleic acid sequences” are those which when aligned andcompared exhibit significant similarities. Standards for homology innucleic acids are either measures for homology generally used in the artby sequence comparison or based upon hybridization conditions, which aredescribed in greater detail below.

Substantial nucleotide sequence homology is observed when there isidentity in nucleotide residues in two sequences (or in theircomplementary strands) when optimally aligned to account for nucleotideinsertions or deletions, in at least about 50%, preferably in at leastabout 75%, more preferably in at least about 90%, and most preferably inat least about 95% of the aligned nucleotides.

Substantial homology also exists when one sequence will hybridize underselective hybridization conditions to another. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 30 nucleotides, preferably at least about65% over a stretch of at least about 25 nucleotides, more preferably atleast about 75%, and most preferably at least about 90% over about 20nucleotides. See, e.g., Kanehisa, Nucleic Acids Res. 12:203 (1984).

The lengths of such homology comparisons may encompass longer stretchesand in certain embodiments may cover a sequence of at least about 17,preferably at least about 25, more preferably at least about 50, andmost preferably at least about 75 nucleotide residues.

Stringency of conditions employed in hybridizations to establishhomology are dependent upon factors such as salt concentration,temperature, the presence of organic solvents, and other parameters.Stringent temperature conditions usually include temperatures in excessof about 30° C., often in excess of about 37° C., typically in excess ofabout 45° C., preferably in excess of about 55° C., more preferably inexcess of about 65° C., and most preferably in excess of about 70° C.Stringent salt conditions will ordinarily be less than about 1000 mM,usually less than about 500 mM, more usually less than about 400 mM,preferably less than about 300 mM, more preferably less than about 200mM, and most preferably less than about 150 mM. For example, saltconcentrations of 100, 50 and 20 mM are used. The combination of theforegoing parameters, however, is more important than the measure of anysingle parameter. See, e.g., Wetmur et al., J. Mol. Biol. 31:349 (1968).

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

The term “substantially pure” is defined herein to mean a mammalianleukotriene receptor, nucleic acid or other material that is free fromother contaminating proteins, nucleic acids, and other biologicalsderived from an original source organism or recombinant DNA expressionsystem. Purity may be assayed by standard methods and will typicallyexceed at least about 50%, preferably at least about 75%, morepreferably at least about 90%, and most preferably at least about 95%purity. Purity evaluation may be made on a mass or molar basis.

Nucleic acids encoding the leukotriene receptors or fragments thereofcan be prepared by standard methods. For example, DNA can be chemicallysynthesized using, e.g., the phosphoramidite solid support method ofMatteucci et al. [J. Am. Chem. Soc. 103:3185 (1981)], the method of Yooet al. [J. Biol. Chem. 764:17078 (1989)], or other well known methods.This can be done by sequentially linking a series of oligonucleotidecassettes comprising pairs of synthetic oligonucleotides, as describedbelow.

Of course, due to the degeneracy of the genetic code, many differentnucleotide sequences can encode the leukotriene receptors. The codonscan be selected for optimal expression in prokaryotic or eukaryoticsystems. Such degenerate variants are of course also encompassed by thisinvention.

Moreover, nucleic acids encoding the leukotriene receptors can readilybe modified by nucleotide substitutions, nucleotide deletions,nucleotide insertions, and inversions of nucleotide stretches. Suchmodifications result in novel DNA sequences that encode antigens havingimmunogenic or antigenic activity in common with the wild-typereceptors. These modified sequences can be used to produce wild type ormutant receptors, or to enhance expression in a recombinant DNA system.

Insertion of the DNAs encoding the leukotriene receptors into a vectoris easily accomplished when the termini of both the DNAs and the vectorcomprise compatible restriction sites. If this cannot be done, it may benecessary to modify the termini of the DNAs and/or vector by digestingback single-stranded DNA overhangs generated by restriction endonucleasecleavage to produce blunt ends, or to achieve the same result by fillingin the single-stranded termini with an appropriate DNA polymerase.

Alternatively, desired sites may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated by the use ofthe polymerase chain reaction (PCR). See, e.g., Saiki et al., Science239:487 (1988). The cleaved vector and the DNA fragments may also bemodified if required by homopolymeric tailing.

Recombinant expression vectors used in this invention are typicallyself-replicating DNA or RNA constructs comprising nucleic acids encodingone of the mammalian leukotriene receptors, usually operably linked tosuitable genetic control elements that are capable of regulatingexpression of the nucleic acids in compatible host cells. Geneticcontrol elements may include a prokaryotic promoter system or aeukaryotic promoter expression control system, and typically include atranscriptional promoter, an optional operator to control the onset oftranscription, transcription enhancers to elevate the level of mRNAexpression, a sequence that encodes a suitable ribosome binding site,and sequences that terminate transcription and translation. Expressionvectors also may contain an origin of replication that allows the vectorto replicate independently of the host cell.

Vectors that could be used in this invention include microbial plasmids,viruses, bacteriophage, integratable DNA fragments, and other vehiclesthat may facilitate integration of the nucleic acids into the genome ofthe host. Plasmids are the most commonly used form of vector but allother forms of vectors which serve an equivalent function and which are,or become, known in the art are suitable for use herein. See, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,Boston, Mass.

Expression of nucleic acids encoding the leukotriene receptors of thisinvention can be carried out by conventional methods in eitherprokaryotic or eukaryotic cells. Although strains of E. coli areemployed most frequently in prokaryotic systems, many other bacteriasuch as various strains of Pseudomonas and Bacillus are know in the artand can be used as well.

Prokaryotic expression control sequences typically used includepromoters, including those derived from the β-lactamase and lactosepromoter systems [Chang et al., Nature, 198:1056 (1977)], the tryptophan(trp) promoter system [Goeddel et al., Nucleic Acids Res. 8:4057(1980)], the lambda P_(L) promoter system [Shimatake et al., Nature,292:128 (1981)] and the tac promoter [De Boer et al., Proc. Natl. Acad.Sci. USA 292:128 (1983)]. Numerous expression vectors containing suchcontrol sequences are known in the art and available commercially.

Suitable host cells for expressing nucleic acids encoding the mammalianleukotriene receptors include prokaryotes and higher eukaryotes.Prokaryotes include both gram negative and positive organisms, e.g., E.coli and B. subtilis. Higher eukaryotes include established tissueculture cell lines from animal cells, both of non-mammalian origin,e.g., insect cells, and birds, and of mammalian origin, e.g., human,primates, and rodents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express themammalian leukotriene receptors include but are not limited to thosecontaining the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipppromoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybridpromoters such as ptac (pDR540). See Brosius et al., “Expression VectorsEmploying Lambda-, trp-, lac-, and Ipp-derived Promoters”, in Rodriguezand Denhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, 1988, Buttersworth, Boston, pp. 205-236.

Higher eukaryotic tissue culture cells are preferred hosts for therecombinant production of the mammalian leukotriene receptors. Althoughany higher eukaryotic tissue culture cell line might be used, includinginsect baculovirus expression systems, mammalian cells are preferred.Transformation or transfection and propagation of such cells have becomea routine procedure. Examples of useful cell lines include HeLa cells,Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) celllines, insect cell lines, bird cell lines, and monkey (COS) cell lines.

Expression vectors for such cell lines usually include an origin ofreplication, a promoter, a translation initiation site, RNA splice sites(if genomic DNA is used), a polyadenylation site, and a transcriptiontermination site. These vectors also usually contain a selection gene oramplification gene. Suitable expression vectors may be plasmids,viruses, or retroviruses carrying promoters derived, e.g., from suchsources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCR®3.1, pCDNA1, pCD [Okayama et al., Mol. Cell Biol. 5:1136(1985)], pMC1neo Poly-A [Thomas et al., Cell 51:503 (1987)], pUC19,pREP8, pSVSPORT and derivatives thereof, and baculovirus vectors such aspAC 373 or pAC 610.

Protein Purification

The proteins, polypeptides and antigenic fragments of this invention canbe purified by standard methods, including but not limited to salt oralcohol precipitation, preparative disc-gel electrophoresis, isoelectricfocusing, high pressure liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, cation and anion exchange and partitionchromatography, and countercurrent distribution. Such purificationmethods are well known in the art and are disclosed, e.g., in Guide toProtein Purification, Methods in Enzymology, Vol. 182, M. Deutscher,Ed., 1990, Academic Press, New York, N.Y. More specific methodsapplicable to purification of the leukotriene receptors are describedbelow.

Purification steps can be followed by carrying out assays for ligandbinding activity as described below. Particularly where a receptor isbeing isolated from a cellular or tissue source, it is preferable toinclude one or more inhibitors of proteolytic enzymes is the assaysystem, such as phenylmethanesulfonyl fluoride (PMSF).

Screening Systems and Methods

The invention allows the discovery of selective agonists and antagonistsof the novel receptor that may be useful in treatment and management ofa variety of diseases including inflammation, asthma, allergy,glomerulonephritis, neuroendocrine dysfunctions, AIDS, arthritis, boweldisease, psoriasis, diabetes, obesity, atherosclerosis, bacterialinfection, encephalomyelitis, etc. Thus, the leukotriene receptor ofthis invention can be employed in screening systems to identify agonistsor antagonists of the receptor. Essentially, these systems providemethods for bringing together a mammalian leukotriene receptor, anappropriate known ligand, including leukotriene itself, and a sample tobe tested for the presence of a leukotriene agonist or antagonist.

Two basic types of screening systems can be used, a labeled-ligandbinding assay and a “functional” assay. A labeled ligand for use in thebinding assay can be obtained by labeling leukotriene or a knownleukotriene agonist or antagonist with a measurable group as describedabove in connection with the labeling of antibodies. Various labeledforms of leukotriene are available commercially or can be generatedusing standard techniques. In an example below, ³H-leukotriene is usedas the ligand.

Typically, a given amount of the leukotriene receptor of the inventionis contacted with increasing amounts of a labeled ligand, such aslabeled leukotriene itself, and the amount of the bound labeled ligandis measured after removing unbound labeled ligand by washing. As theamount of the labeled ligand is increased, a point is eventually reachedat which all receptor binding sites are occupied or saturated. Specificreceptor binding of the labeled ligand is abolished by a large excess ofunlabeled ligand.

Preferably, an assay system is used in which non-specific binding of thelabeled ligand to the receptor is minimal. Non-specific binding istypically less than 50%, preferably less than 15%, and more preferablyless than 10% of the total binding of the labeled ligand.

As used herein, the term “leukotriene ligand” is defined to meanleukotriene itself or an analog of leukotriene, and extending up to thecomplete leukotriene molecule. For regulatory purposes it may bedesirable to use leukotriene or an active fragment thereof as theleukotriene ligand in conjunction with the human receptor when screeningfor leukotriene agonists or antagonists for human therapeutic purposes.

In principle, a binding assay of the invention could be carried outusing a soluble receptor of the invention, e.g., following productionand refolding by standard methods from an E. coli expression system, andthe resulting receptor-labeled ligand complex could be precipitated,e.g., using an antibody against the receptor. The precipitate could thenbe washed and the amount of the bound labeled ligand could be measured.

Preferably, however, a nucleic acid encoding one of the leukotrienereceptors of the invention is transfected into an appropriate host cell,whereby the receptor will become incorporated into the membrane of thecell. A membrane fraction can then be isolated from the cell and used asa source of the receptor for assay. Preferably, specific binding of thelabeled ligand to a membrane fraction from the untransfected host cellwill be negligible.

The binding assays of this invention can be used to identify bothleukotriene agonists and antagonists, because both will interfere withthe binding of the labeled ligand to the receptor.

In the basic binding assay, the method for identifying a leukotrieneagonist or antagonist comprises:

-   -   (a) contacting a mammalian leukotriene receptor having an amino        acid sequence defined by SEQ ID NO: 2 or a subsequence thereof,        in the presence of a known amount of labeled leukotriene with a        sample to be tested for the presence of a leukotriene agonist or        antagonist; and    -   (b) measuring the amount of labeled leukotriene bound to the        receptor;

whereby a leukotriene agonist or antagonist in the sample is identifiedby measuring substantially reduced binding of the labeled leukotriene tothe leukotriene receptor, compared to what would be measured in theabsence of such agonist or antagonist.

Preferably, the leukotriene receptor used to identify a leukotrieneagonist or antagonist for human therapeutic purposes has an amino acidsequence defined by SEQ ID NO: 2 or a subsequence thereof.

In one embodiment of the invention, the foregoing method furthercomprises:

-   -   (c) Contacting a mammalian leukotriene receptor in the presence        of a known amount of labeled leukotriene with a compound        identified as a leukotriene agonist or antagonist in steps (a)        and (b); and    -   (d) Measuring the amount of labeled leukotriene bound to the        receptor;        whereby a leukotriene agonist or antagonist specific for the        leukotriene receptor is identified by measuring substantially        undiminished binding of the labeled leukotriene to the receptor,        compared to what would be measured in the absence of such        agonist or antagonist.

Determination of whether a particular molecule inhibiting binding of thelabeled ligand to the receptor is an antagonist or an agonist is thendetermined in a second, functional assay. The functionality ofleukotriene agonists and antagonists identified in the binding assay canbe determined in cellular and animal models.

Functional Assays for Antagonists/Agonists of Leukotriene Receptors

In cellular models, parameters for intracellular activities mediated byleukotriene receptors can be monitored for antagonistic and/or agonisticactivities. Such parameters include but are not limited to intracellularsecond messenger pathways activated via the leukotriene receptors,changes in cell growth rate, secretion of hormones, etc., usingpublished methods. Examples of such methods are, measurement of theeffects of the ligands on receptor-mediated inhibition offorskolin-stimulated intracellular cAMP production [Parker et al., Mol.Brain Res. 34:179-189 (1995)], receptor-stimulated Ca²⁺⁺ mobilizationand mitogenic effects [Sethi et al., Cancer Res. 51:1674-1679 (1991)],and inositol phosphate production and MAP kinase induction (Wang et al.,Biochemistry 37:6711-17 (1998). The FLIPR method described in thisinvention is also suitable for measuring intracellular release ofcalcium.

Agonists of leukotriene receptors may also be identified directly byusing functional assays. An agonist may or may not directly inhibitleukotriene binding to leukotriene receptors.

In addition to the methods described above, activities of an antagonistmay be measured in cellular models for altered intracellular cAMP orCa²⁺ concentrations (Yokomizo et al. Nature, 1997, 387:620).Leukotriene-induced chemotaxis using cultured cells can also be utilized(Yokomizo et al. Nature, 1997, 387:620). Furthermore, models employingXenopus laevis, pigment dispersion/aggregation in melanophores, andaequorin assay in mammalian cells are suitable for this purpose (Lynchet al., Nature, 1999, 399:790). Methods using animals or animal tissuesfor such activities can also be employed. Leukotriene-stimulatedneutrophil chemotaxis (Palmer et al. Prostaglandins, 1980, 20:411-418),enhanced neutrophil-endothelial interaction (Hoover et al. Proc. Natl.Acad. Sci. U.S.A., 1984, 81:2191-2193), neutrophil activation leading todegranulation and release of mediators, enzymes and superoxides (Sha'afiet al. J. Cell Physiol. 1981, 108:401-408), inflammatory pain (Levine etal. Science, 1984, 225:743-745), and increased cytokine production(Brach et al. Eur. J. Immunol. 1992, 22:2705-2711) and transcription(Stanova, et al. Biochem. J. 1992, 282:625-629) are examples of suchmethods.

Other Mammalian Leukotriene Receptors

The present invention provides methods for cloning mammalian leukotrienereceptors from other mammalian species. Briefly, Southern and Northernblot analysis can be carried out to identify cells from other speciesexpressing genes encoding the leukotriene receptors. Complementary DNA(cDNA) libraries can be prepared by standard methods from mRNA isolatedfrom such cells, and degenerate probes or PCR primers based on thenucleic acid and amino acid sequences provided herein can be used toidentify clones encoding a leukotriene receptor.

Alternatively, expression cloning methodology can be used to identifyparticular clones encoding a leukotriene receptor. An antibodypreparation which exhibits cross-reactivity with leukotriene receptorsfrom a number of mammalian species may be useful in monitoringexpression cloning.

However identified, clones encoding leukotriene receptors from variousmammalian species can be isolated and sequenced, and the coding regionscan be excised and inserted into an appropriate vector.

Other Related Genes

The present invention also provides compositions and methods for cloningother genes related to the gene encoding a polypeptide defined by SEQ IDNO: 2. Specifically, this invention provides a recombinant nucleic acidcomprising a sequence having at least about 70% identity over a stretchof at least about 30 nucleotides to the nucleic acid sequence of SEQ IDNO: 1, useful, e.g., as a probe or PCR primer for a related gene.

Localization of mRNA encoding the polypeptide of SEQ ID NO: 2

The present invention also provides compositions and methods forlocalization of messenger RNA coding for the polypeptide defined by theamino acid sequence of SEQ ID NO: 2.

Specifically, human multiple tissue and cancer cell line blotscontaining approximately 2 μg of poly(A)⁺ RNA per lane, are purchasedfrom Clontech (Palo Alto, Calif.). Probes are radiolabeled with [α-³²P]dATP, e.g., using the Amersham Rediprime random primer labeling kit(RPN1633). Prehybridization and hybridizations are performed at 65° C.in 0.5M Na₂HPO₄, 7% SDS, 0.5M EDTA (pH 8.0). High stringency washes areconducted, e.g., at 65° C. with two initial washes in 2×SSC, 0.1% SDSfor 40 min followed by a subsequent wash in 0.1×SSC, 0.1% SDS for 20min.Membranes are then exposed at −70° C. to X-Ray film (Kodak) in thepresence of intensifying screens. More detailed studies by cDNA librarySoutherns are performed with selected clones of nucleic acids having thenucleotide sequence defined by SEQ ID NO: 1 to examine their expressionin other cell subsets.

Two prediction algorithms that take advantage of the patterns ofconservation and variation in multiply aligned sequences, (Rost andSander (1994) Proteins 19:55-72) and DSC (King and Sternberg (1996)Protein Sci. 5:2298-2310), are used.

Alternatively, two appropriate primers are selected and RT-PCR is usedon an appropriate mRNA sample selected for the presence of message toproduce a cDNA, e.g., a sample which expresses the gene.

Full length clones may be isolated by hybridization of cDNA librariesfrom appropriate tissues pre-selected by PCR signal.

Message for genes encoding a polypeptide having the amino acid sequenceof SEQ ID NO: 2 are assayed by appropriate technology, e.g., PCR,immunoassay, hybridization, or otherwise. Tissue and organ cDNApreparations are available, e.g., from Clontech, Mountain View, Calif.

Southern Analysis on cDNA libraries are performed as follows: DNA (5 μg)from a primary amplified cDNA library is digested with appropriaterestriction enzymes to release the inserts, run on a 1% agarose gel andtransferred to a nylon membrane (Schleicher and Schuell, Keene, N.H.).

Samples for human mRNA isolation may include, e.g.: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcells CD4+CD45RO- T cells polarized 27 days in anti-CD28, IL-4, and antiIFN-γ, TH2 polarized, activated with anti-CD3 and anti-CD28 4h (T116); Tcell tumor lines Jurkat and Hut78, resting (T117); T cell clones, pooledAD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T cellrandom γδT cell clones, resting (T119); Splenocytes, resting (B100);Splenocytes, activated with anti-CD40 and IL-4 (B 101); B cell EBV linespooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102); B cellline JY, activated with PMA and ionomycin for 1, 6 h pooled (B103); NK20 clones pooled, resting (K100); NK 20 clones pooled, activated withPMA and ionomycin for 6 h (K101); NKL clone, derived from peripheralblood of LGL leukemia patient, IL-2 treated (K106); NK cytotoxic clone640-A30-1, resting (K107); hematopoietic precursor line TF1, activatedwith PMA and ionomycin for 1, 6 h pooled (C100); U937 premonocytic line,resting (M100); U937 premonocytic line, activated with PMA and ionomycinfor 1, 6 h pooled (M101); elutriated monocytes, activated with LPS,IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102); elutriatedmonocytes, activated with LPS, IFNγ, IL-10 for 1, 2, 6, 12, 24 h pooled(M103); elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for4, 16 h pooled (M106); elutriated monocytes, activated with LPS, IFNγ,IL-10 for 4, 16 h pooled (M107); elutriated monocytes, activated LPS for1 h (M108); elutriated monocytes, activated LPS for 6 h (M109); DC 70%CD1a+, from CD34+GM-CSF, TNFα 12 days, resting (D101); DC 70% CD1a+,from CD34+GM-CSF, TNFα 12 days, activated with PMA and ionomycin for 1hr (D102); DC 70% CD1a+, from CD34+GM-CSF, TNFα 12 days, activated withPMA and ionomycin for 6 hr (D103); DC 95% CD1a+, from CD34+GM-CSF, TNFα12 days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled(D104); DC 95% CD14+, ex CD34+GM-CSF, TNFα 12 days FACS sorted,activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CD1a+CD86+,from CD34+GM-CSF, TNFα 12 days FACS sorted, activated with PMA andionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5days, resting (D107); DC from monocytes GM-CSF, IL-4 5 days, resting(D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 hpooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFα,monocyte supernatant for 4, 16 h pooled (D110); leiomyoma L11 benigntumor (X101); normal myometrium M5 (O115); malignant leiomyosarcoma GS1(X103); lung fibroblast sarcoma line MRC5, activated with PMA andionomycin for 1, 6 h pooled (C101); kidney epithelial carcinoma cellline CHA, activated with PMA and ionomycin for 1, 6 h pooled (C102);kidney fetal 28 wk male (O100); lung fetal 28 wk male (O101); liverfetal 28 wk male (O102); heart fetal 28 wk male (O103); brain fetal 28wk male (O104); gallbladder fetal 28 wk male (O106); small intestinefetal 28 wk male (O107); adipose tissue fetal 28 wk male (O108); ovaryfetal 25 wk female (O109); uterus fetal 25 wk female (O110); testesfetal 28 wk male (O111); spleen fetal 28 wk male (O112); adult placenta28 wk (O113); tonsil inflamed, from 12 year old (X100); psoriasis humanskin sample; normal human skin sample; pool of rheumatoid arthritishuman; Hashimoto's thyroiditis thyroid; normal human thyroid; ulcerativecolitis human colon; normal human colon; normal weight monkey colon;pheumocystic carnii pneumonia lung; allergic lung; poll of three heavysmoker human lung; pool of two normal human lung; Ascaris-challengedmonkey lung, 24 hr; Ascaris-challenged monkey lung, 4hr; and normalweight monkey lung.

Antibody Production

Antigenic (i.e., immunogenic) fragments of the mammalian leukotrienereceptors of this invention, which may or may not have ligand bindingactivity, may be produced. Regardless of whether they bind leukotriene,such fragments, like the complete receptors, are useful as antigens forpreparing antibodies by standard methods that can bind to the completereceptors. Shorter fragments can be concatenated or attached to acarrier. Because it is well known in the art that epitopes generallycontain at least about five, preferably at least about 8, amino acidresidues [Ohno et al., Proc. Natl. Acad. Sci. USA 82:2945 (1985)],fragments used for the production of antibodies will generally be atleast that size. Preferably, they will contain even more residues, asdescribed above. Whether a given fragment is immunogenic can readily bedetermined by routine experimentation.

Although it is generally not necessary when complete mammalianleukotriene receptors are used as antigens to elicit antibody productionin an immunologically competent host, smaller antigenic fragments arepreferably first rendered more immunogenic by cross-linking orconcatenation, or by coupling to an immunogenic carrier molecule (i.e.,a macromolecule having the property of independently eliciting animmunological response in a host animal). Cross-linking or conjugationto a carrier molecule may be required because small polypeptidefragments sometimes act as haptens (molecules which are capable ofspecifically binding to an antibody but incapable of eliciting antibodyproduction, i.e., they are not immunogenic). Conjugation of suchfragments to an immunogenic carrier molecule renders them moreimmunogenic through what is commonly known as the “carrier effect”.

Suitable carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides etc. Protein carrier molecules are especiallypreferred, including but not limited to keyhole limpet hemocyanin andmammalian serum proteins such as human or bovine gammaglobulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, but not necessarily, the protein carrier will beforeign to the host animal in which antibodies against the fragments areto be elicited.

Covalent coupling to the carrier molecule can be achieved using methodswell known in the art, the exact choice of which will be dictated by thenature of the carrier molecule used. When the immunogenic carriermolecule is a protein, the fragments of the invention can be coupled,e.g., using water-soluble carbodiimides such as dicyclohexylcarbodiimideor glutaraldehyde.

Coupling agents such as these can also be used to cross-link thefragments to themselves without the use of a separate carrier molecule.Such cross-linking into aggregates can also increase immunogenicity.Immunogenicity can also be increased by the use of known adjuvants,alone or in combination with coupling or aggregation.

Suitable adjuvants for the vaccination of animals include but are notlimited to Adjuvant 65 (containing peanut oil, mannide monooleate andaluminum monostearate); Freund's complete or incomplete adjuvant;mineral gels such as aluminum hydroxide, aluminum phosphate and alum;surfactants such as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The polypeptides could also be administered following incorporation intoliposomes or other microcarriers.

Information concerning adjuvants and various aspects of immunoassays aredisclosed, e.g., in the series by P. Tijssen, Practice and Theory ofEnzyme Immunoassays, 3rd Edition, 1987, Elsevier, N.Y. Other usefulreferences covering methods for preparing polyclonal antisera includeMicrobiology, 1969, Hoeber Medical Division, Harper and Row;Landsteiner, Specificity of Serological Reactions, 1962, DoverPublications, New York, and Williams, et al., Methods in Immunology andImmunochemistry, Vol. 1, 1967, Academic Press, New York.

Serum produced from animals immunized using standard methods can be useddirectly, or the IgG fraction can be separated from the serum usingstandard methods such as plasmaphoresis or adsorption chromatographywith IgG-specific adsorbents such as immobilized Protein A.Alternatively, monoclonal antibodies can be prepared.

Hybridomas producing monoclonal antibodies against the leukotrienereceptors of the invention or antigenic fragments thereof are producedby well-known techniques. Usually, the process involves the fusion of animmortalizing cell line with a B-lymphocyte that produces the desiredantibody. Alternatively, non-fusion techniques for generating immortalantibody-producing cell lines can be used, e.g., virally-inducedtransformation [Casali et al., Science 234:476 (1986)]. Immortalizingcell lines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine, and human origin. Most frequently, rat or mousemyeloma cell lines are employed as a matter of convenience andavailability.

Techniques for obtaining antibody-producing lymphocytes from mammalsinjected with antigens are well known. Generally, peripheral bloodlymphocytes (PBLs) are used if cells of human origin are employed, orspleen or lymph node cells are used from non-human mammalian sources. Ahost animal is injected with repeated dosages of the purified antigen(human cells are sensitized in vitro), and the animal is permitted togenerate the desired antibody-producing cells before they are harvestedfor fusion with the immortalizing cell line. Techniques for fusion arealso well known in the art, and in general involve mixing the cells witha fusing agent, such as polyethylene glycol.

Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Those secreting thedesired antibody are selected using standard immunoassays, such asWestern blotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), or the like. Antibodies are recovered from themedium using standard protein purification techniques [Tijssen, Practiceand Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)].

Many references are available to provide guidance in applying the abovetechniques [Kohler et al., Hybridoma Techniques (Cold Spring HarborLaboratory, New York, 1980); Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal AntibodyTechnology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal HybridomaAntibodies: Techniques and Applications (CRC Press, Boca Raton, Fla.,1982)]. Monoclonal antibodies can also be produced using well-knownphage library systems. See, e.g., Huse, et al., Science 246:1275 (1989);Ward, et al., Nature, 341:544 (1989).

Antibodies thus produced, whether polyclonal or monoclonal, can be used,e.g., in an immobilized form bound to a solid support by well knownmethods, to purify the receptors by immunoaffinity chromatography.

Antibodies against the antigenic fragments can also be used, unlabeledor labeled by standard methods, as the basis for immunoassays of themammalian leukotriene receptors. The particular label used will dependupon the type of immunoassay. Examples of labels that can be usedinclude but are not limited to radiolabels such as ³²P, ¹²⁵I, ³H and¹⁴C; fluorescent labels such as fluorescein and its derivatives,rhodamine and its derivatives, dansyl and umbelliferone;chemiluminescers such as luciferia and 2,3-dihydrophthalazinediones; andenzymes such as horseradish peroxidase, alkaline phosphatase, lysozymeand glucose-6-phosphate dehydrogenase.

The antibodies can be tagged with such labels by known methods. Forexample, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bisdiazotized benzadine and the like may be usedto tag the antibodies with fluorescent, chemiluminescent or enzymelabels. The general methods involved are well known in the art and aredescribed, e.g., in Immunoassay: A Practical Guide, 1987, Chan (Ed.),Academic Press, Inc., Orlando, Fla. Such immunoassays could be carriedout, for example, on fractions obtained during purification of thereceptors.

The antibodies of the present invention can also be used to identifyparticular cDNA clones expressing the leukotriene receptors inexpression cloning systems.

Neutralizing antibodies specific for the ligand-binding site of areceptor can also be used as antagonists (inhibitors) to blockleukotriene binding. Such neutralizing antibodies can readily beidentified through routine experimentation, e.g., by using theradioligand binding assay described infra. Antagonism of leukotrieneactivity can be accomplished using complete antibody molecules, orwell-known antigen binding fragments such as Fab, Fc, F(ab)₂, and Fvfragments.

Definitions of such fragments can be found, e.g., in Klein, Immunology(John Wiley, New York, 1982); Parham, Chapter 14, in Weir, ed.Immunochemistry, 4th Ed. (Blackwell Scientific Publishers, Oxford,1986). The use and generation of antibody fragments has also beendescribed, e.g.: Fab fragments [Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985)], Fv fragments [Hochman et al.,Biochemistry 12:1130 (1973); Sharon et al., Biochemistry 15:1591 (1976);Ehrlich et al., U.S. Pat. No. 4,355,023] and antibody half molecules(Auditore-Hargreaves, U.S. Pat. No. 4,470,925). Methods for makingrecombinant Fv fragments based on known antibody heavy and light chainvariable region sequences have further been described, e.g., by Moore etal. (U.S. Pat. No. 4,642,334) and by Plückthun [Bio/Technology 9:545(1991)]. Alternatively, they can be chemically synthesized by standardmethods.

Anti-idiotypic antibodies, both polyclonal and monoclonal, can also beproduced using the antibodies elicited against the receptors asantigens. Such antibodies can be useful as they may mimic the receptors.

Pharmaceutical Compositions

The leukotriene receptor agonists and antagonists of this invention canbe used therapeutically to stimulate or block the activity ofleukotriene, and thereby to treat any medical condition caused ormediated by leukotriene. The dosage regimen involved in a therapeuticapplication will be determined by the attending physician, consideringvarious factors which may modify the action of the therapeuticsubstance, e.g., the condition, body weight, sex and diet of thepatient, the severity of any infection, time of administration, andother clinical factors.

Typical protocols for the therapeutic administration of such substancesare well known in the art. Administration of the compositions of thisinvention is typically by parenteral, by intraperitoneal, intravenous,subcutaneous, or intramuscular injection, or by infusion or by any otheracceptable systemic method. Often, treatment dosages are titrated upwardfrom a low level to optimize safety and efficacy. Generally, dailydosages will fall within a range of about 0.01 to 20 mg protein perkilogram of body weight. Typically, the dosage range will be from about0.1 to 5 mg per kilogram of body weight.

Dosages will be adjusted to account for the smaller molecular sizes andpossibly decreased half-lives (clearance times) followingadministration. It will be appreciated by those skilled in the art,however, that the leukotriene antagonists of the invention encompassneutralizing antibodies or binding fragments thereof in addition toother types of inhibitors, including small organic molecules andinhibitory ligand analogs, which can be identified using the methods ofthe invention.

An “effective amount” of a composition of the invention is an amountthat will ameliorate one or more of the well-known parameters thatcharacterize medical conditions caused or mediated by leukotriene.

Although the compositions of this invention could be administered insimple solution, they are more typically used in combination with othermaterials such as carriers, preferably pharmaceutical carriers. Usefulpharmaceutical carriers can be any compatible, non-toxic substancessuitable for delivering the compositions of the invention to a patient.Sterile water, alcohol, fats, waxes, and inert solids may be included ina carrier. Pharmaceutically acceptable adjuvants (buffering agents,dispersing agents) may also be incorporated into the pharmaceuticalcomposition. Generally, compositions useful for parenteraladministration of such drugs are well known; e.g. Remington'sPharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, Pa.,1990). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems [Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)].

Therapeutic formulations may be administered in many conventional dosageformulation. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers. Formulations may include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. See,e.g., Gilman et al. (eds.) (1990), The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, supra, Easton, Pa.; Avis et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications Dekker, New York; Lieberman et al.(eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; andLieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: DisperseSystems Dekker, New York.

The present invention also encompasses anti-idiotypic antibodies, bothpolyclonal and monoclonal, which are produced using the above-describedantibodies as antigens. These antibodies are useful because they maymimic the structures of the receptors.

Anti-sense Molecules

The present invention also encompasses anti-sense oligonucleotidescapable of specifically hybridizing to mRNA encoding a mammalianleukotriene receptor having an amino acid sequence defined by SEQ ID NO:2 or a subsequence thereof so as to prevent translation of the mRNA.Additionally, this invention contemplates anti-sense oligonucleotidescapable of specifically hybridizing to the genomic DNA molecule encodinga mammalian leukotriene receptor having an amino acid sequence definedby SEQ ID NO: 2 or a subsequence thereof.

This invention further provides pharmaceutical compositions comprising(a) an amount of an oligonucleotide effective to reduce activity ofhuman leukotriene receptor by passing through a cell membrane andbinding specifically with mRNA encoding human leukotriene receptor inthe cell so as to prevent its translation and (b) a pharmaceuticallyacceptable carrier capable of passing through a cell membrane. In anembodiment, the oligonucleotide is coupled to a substance thatinactivates mRNA. In another embodiment, the substance that inactivatesmRNA is a ribozyme.

EXAMPLES

The present invention can be illustrated by the following examples.Unless otherwise indicated, percentages given below for solids in solidmixtures, liquids in liquids, and solids in liquids are on a wt/wt,vol/vol and wt/vol basis, respectively. Sterile conditions weregenerally maintained during cell culture.

Materials and General Methods

Human marathon-ready cDNAs and RACE kit were from Clontech.Oligonucleotides were custom-synthesized by Gibco Life Technologies.293-EBNA cell line was obtained from Invitrogen. Leukotrienes and otherligands were purchased from Sigma Chemicals. Radioligands were from NEN.

Standard methods were used, as described, e.g., in Maniatis et al.,Molecular Cloning: A Laboratory Manual, 1982, Cold Spring HarborLaboratory, Cold Spring Harbor Press; Sambrook et al., MolecularCloning: A Laboratory Manual, (2d ed.), Vols 1-3, 1989, Cold SpringHarbor Press, N.Y.; Ausubel et al., Biology, Greene PublishingAssociates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements),Current Protocols in Molecular Biology, Greene/Wiley, New York; Innis etal. (eds.) PCR Protocols: A Guide to Methods and Applications, 1990,Academic Press, N.Y.

Example 1 Cloning and Characterization of the Human Leukotriene Receptor

The amino acid sequences of known G-protein coupled receptors were usedto conduct a BLAST search of EST databases. The search identified a 397bp EST as being a putative G-protein coupled receptor fragment. Aphylogenetic analysis (Wisconsin Package, Genetics Computer Group,Madison, Wis.) of this EST suggested that the sequence could be afragment of a leukotriene receptor cDNA. The complete coding region ofthe cDNA was assembled by combining contiguous sequences from DNAdatabases and RACE PCR.

To clone the full length cDNA(referred to hereinafter as SP9030), a PCRprimer pair (oligo 358 and oligo 359) was designed, based on theassembled sequences, to amplify in a PCR using Clontech marathon-readycDNA from liver as template. The PCR product containing the full lengthcDNA (1.1-kb) was cloned into expression vector pCR3.1 (Invitrogen) toform an expression construct pCR3.1-SP9030. PCR conditions for this PCRwas 94° C. for 30 sec; 35 cycles at 94° C. for 30sec, 65° C. for 30 sec,72° C. for 2 min; and 72° C. for 7 min. The sequences of this pair ofPCR primers are: Oligo 358, gccgccaccatgtcggtctgctaccgtcc (SEQ ID NO: 3)and Oligo 359, gcaggttgtagggtctgctgtca (SEQ ID NO: 4). Sequencinganalysis identified an open reading frame of 1077 bp (SEQ ID NO: 1) thatencodes a protein of 358 amino acids (aa) (SEQ ID NO: 2).

Hydrophobicity analysis of the 358aa protein suggests that there areseven transmembrane spanning regions within the protein, a feature thatis shared by the G-protein coupled receptor super-family. BLAST analysiswith the amino acids of SP9030 against the Genbank database revealedhigh homology to three known receptors, i.e. the human leukotriene B4receptor (Yokomizo et al., Nature, 387:620 (1997)), the human CRTH12(Nagata et al., J. Immunol., 162:1278-1286 (1999)), and the humansomatostatin receptor SSTR4 (Rohrer et al., Proc. Natl. Acad. Sci. USA.,90(9):4196-4200 (1993)). Sequence alignment analysis of the proteinsequences using the J. Hein method (Guide to Protein Purification,Methods in Enzymology, 183:626-645 (1990), M. Deutscher, Ed., 1990,Academic Press, New York, N.Y.) showed highest homology of SP9030 toleukotriene B4 receptor (42%), followed by CRTH12 (32%) and SSTR4 (27%).These analyses suggest that the protein encoded by SP9030 ORF may be aleukotriene receptor.

In addition to the high homology of SP9030 to leukotriene B4 receptor(Yokomizo et al., Nature, 387:620-624 (1997)) (accession #D89079),physical linkage was observed between the two genes. The 5′ untranslatedregion of leukotriene B4 receptor (accession #D89079) is identical tothe coding region at the 3′ end of the ORF and the immediate down stream3′ untranslated sequence. This analysis indicates that the two receptorscan exist on a single messenger RNA and thus be co-expressed in thecell.

Example 2 Agonist/antagonist Screening Assay

Transfection of Cells and Membrane Preparations

COS-7 cells, grown in DMEM/10% FCS until 80-90% confluence, weretransfected with SuperFect agent (Giagene) at 20 μg DNA/150 mm plate.Forty eight hours after transfection, medium was changed to Opti-MEM orDMEM/Opti-MEM(1:1)/5% FCS. Cells were harvested 72 hours aftertransfection and membranes from the cells were prepared as follows. Thecells were washed with 20 ml PBS without Ca²⁺/Mg²⁺ and incubated with 10ml 10 nM Hepes pH 7.4, 0.5 mM PMSF, 20 μg/ml aprotinin. The cells werescraped off the plate and vortexed. The cell suspension was thencentrifuged at 13,000 g at 4° C. for 15 min. The pellets werere-suspended in 1.8 ml 50 mM Tris-Cl, pH 7.5 and vortexed. The membraneswere homogenized with a 23-gauge needle. The protein concentration ofthe membrane preparations was determined with the BCA agents (Pierce).

Radioligand Binding Assay

Radioligand binding assays were performed to test the ability of SP9030,when expressed in cultured cells, to bind ³H-labeled leukotriene. TheORF of SP9030 was cloned in the expression vector pCR3.1(pCR3.1-SP9030). COS-7 cells were transfected with pCR3.1-SP9030 orpCR3.1 alone (mock transfection). Two days after transfection, thenormal growth medium DMEM/10% FCS was replaced by either Opti-MEM orDMEM-Opti-MEM/5% FCS. The cells were allowed to grow one more day andthen membranes were prepared for use in binding assay. Unlabeled LTB4 at1 μM was used to determine non-specific binding. After a total of threedays from transfection, membranes were prepared from the transfectantcells and specific binding to ³H-LTB4 observed.

For saturation binding, 150 μl binding assay buffer (30 mM Hepes, pH7.4, 10 mM CaCl₂, 10 mM MgCl₂, 0.05% fatty acid-free BSA (w/v), keptcold on ice) containing 24 μg of membranes were mixed with 50 μl ofbinding assay buffer containing 2% (v/v) DMSO cold leukotriene (1 μM).³H-LTB4/ethanol (NEN, 50 nM) was added to the assays at increasingconcentrations. The reactions were incubated for 1 hour at 4° C. whilerotated slowly. Multiscreen FB filters (Millipore) pre-soaked with 50 μlbinding assay buffer for 1 hour at room temperature were used to filterthe binding assays and the filters were washed twice with 100 μl 50 mMTrisCl, pH 7.5 (ice cold). Fifty microliters of scintillation fluid wasadded to the filters and counted to detect the bound radioligands. Forradioligand competition assays, 160 μl binding assay buffer containingappropriate membranes were mixed with 20 μl of binding assay buffercontaining 6% DMSO (v/v) and various concentrations of competingcompounds. A final 20 μl of binding assay buffer containing 6% (v/v)DMSO and 1 μl of ³H-LTB4/ethanol (NEN, 50 nM) was added to start thebinding reaction. The final concentration of radioligand was 0.25 nM.Incubation conditions were the same as that used for the above describedsaturation assays.

Saturation radioligand binding assays yielded a K_(D) of 0.17±0.07 nMand B_(max) of 70±8 fmol/mg (n=3). Radioligand competition assays using³H-LTB4 as the label was employed to test the affinities of 13 unlabeledleukotriene and lipoxin compounds. COS-7 cells transfected with SP9030cDNA were harvested three days after transfection and membrane preparedfrom cells used for binding assays. An unlabeled leukotriene or lipoxinat increasing concentration was used to compete with ³H-leukotriene B4(0.25 nM). K_(i) values are calculated for individual compound by usingKi=EC₅₀/(1+[³H-LTB4]/Kd), where [³H-LTB4] is the concentration of theradioligand used in the assay (0.25 nM), K_(D) is the affinity of theradioligand for the receptor (0.17 nM) and EC₅₀ is determined bynon-linear regression analysis. High affinity for LTB4, LTB5 and lipoxinA4 were revealed (Ki=2.0±1.2, 8.6±5.3 and 112±40 nM, respectively). Theother compounds displayed substantially weaker or no affinities forSP9030 (Ki>1 μM). These results indicate that the receptor encoded bySP9030 is structurally selective for certain ligands.

Intracellular Ca²⁺ concentration measurement

293-EBNA cells, grown in DMEM containing 10% FCS until 80-90%confluence, were transfected with SuperFect transfection agent. The nextday, cells were trypsinized off culture plates and washed with PBSlacking Ca²⁺/Mg²⁺. The cells were then seeded at a density of 35,000cells per 100 μl medium) into 96-well plates that were pre-coated withpoly-D-lysine (Becton Dickinson). The third day following transfection,medium was removed from cells and 100 μl Hank's balanced salt solution(lacking phenol red) containing 4 μM of Fluo-3, AM (Molecular Probes),20 mM Hepes, pH 7.4, 0.1% (w/v) BSA and 250 mM probenecid added andsubsequently incubated at 37° C., 5% CO₂ for 1 hour. The cells were thenwashed three times with 150 μl wash buffer containing HANK's BSS, 40 mMHepes, pH 7.4 and 250 mM probenecid. One hundred μl of the wash bufferwas added after the final wash and Ca²⁺ flux was measured after additionof 40 μl of wash buffer containing appropriate concentration of ligands.The FLIPR instrument (Molecular Device) was used in the measurement ofCa²⁺ flux.

Intracellular function that the SP9030 receptor may mediate throughactivation by its ligand was examined in measurements of intracellularCa²⁺ flux. Interaction of SP9030 expressed in 293-EBNA cells with LTB4activated cellular Ca²⁺ release, suggesting SP9030 is able to stimulatecellular functions by coupling to G protein(s) in the cell. Cells thatwere mock transfected without SP9030 cDNA did not respond to incubationwith LTB4.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, together with the full scope ofequivalents to which such claims are entitled.

1. An isolated polypeptide comprising at least 12 contiguous residues ofthe amino acid sequence of SEQ ID NO:
 2. 2. The polypeptide of claim 1comprising at least 20 contiguous residues of the amino acid sequence ofSEQ ID NO:
 2. 3. The polypeptide of claim 1 comprising at least 30contiguous residues of the amino acid sequence of SEQ ID NO:
 2. 4. Anisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2.5. The polypeptide of claim 4 which is bound to leukotriene B4.
 6. Thepolypeptide of claim 5 wherein the leukotriene B4 is labeled with ³H. 7.A method for identifying an agonist or antagonist of a mammalianleukotriene receptor, comprising: (a) contacting a polypeptide of claim4 in the presence of a known amount of labeled leukotriene with a sampleto be tested for the presence of a leukotriene agonist or antagonist;and (b) measuring the amount of labeled leukotriene specifically boundto the polypeptide; whereby a leukotriene agonist or antagonist in thesample is identified by measuring substantially reduced binding of thelabeled leukotriene to the polypeptide, compared to what would bemeasured in the absence of such agonist or antagonist.
 8. The method ofclaim 7 in which membranes isolated from mammalian cells comprising anucleic acid encoding the polypeptide are used as the source of saidpolypeptide.